分枝杆菌ESX-1分泌系统调节蛋白MycP1的结构生物学研究
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摘要
结核病是目前世界范围内流行范围最广、致死率最高的的传染性疾病之一。全球每年新增近千万感染者,并造成近百万人死亡。结核分枝杆菌是结核病的致病菌,它能够产生多种致病因子,具有强致病性；同时具有多重耐药性和免疫逃逸能力,对寄生环境有极强的适应能力。因此,结核分枝杆菌一直都是对人类健康威胁最大的致病菌之一。近年来,对结核分枝杆菌致病性和耐药性的研究取得了较大的进展,越来越多与之相关的蛋白被发现。在本论文中,我们对分枝杆菌致病因子分泌系统ESX-1的调节蛋白MycP1以及结核分枝杆菌外膜蛋白RV1698开展结构生物学研究,用X-射线晶体衍射方法解析了它们的三维结构,并探究了结构与功能之间的关系,初步阐释了这两个蛋白质在分枝杆菌致病过程中的作用机制。
本论文的第一部分是分枝杆菌ESX-1分泌系统调节蛋白MycP1的结构生物学研究。结核分枝杆菌中存在一种新型分泌系统T7SS (Type Ⅶ secretion system,), ESX-1系统就是一种典型的T7SS,也是分枝杆菌中研究最广泛的分泌系统。ESX-1分泌系统是分枝杆菌致病性的决定因素之一,致病性分枝杆菌在感染宿主细胞的过程中会通过ESX-1系统分泌致病性蛋白ESAT-6和CFP-10,通过抑制巨噬细胞内吞噬小体的成熟和细胞因子信号、抑制Toll样受体信号通路、在吞噬小体表面形成通道以促进细菌扩散等作用机制促进病菌侵染过程。MycP1是ESX-1系统的组成蛋白之一,对ESX-1系统有双重调控作用：一方面MycPl是维持ESX-1系统完整性所必须的,它的缺失会导致ESX-1系统丧失对ESAT-6/CFP-10的分泌功能；另一方面,MycP1对ESX-1系统的活性具有负调控作用,MycP1活性的缺失可以促进ESX-1系统对ESAT-6/CFP-10的分泌。MycP1的双重调节作用的意义可能在于维持分枝杆菌致病性与免疫原性之间的平衡,避免细菌在侵染宿主细胞的过程中激发宿主过强免疫反应。实际上,MycP1是一种subtilisin-like的丝氨酸蛋白酶,具有跨膜结构,研究证实MycP1对ESX-1系统的负调控是通过降解其天然底物EspB实现的。我们所感兴趣的是,MycP1蛋白作为一个具有双重调控作用的丝氨酸蛋白酶,它在结构上有没有特殊性,在功能上与EspB蛋白相互作用的机制是什么,它又是如何行使双重调控功能的。我们成功地解析了耻垢分枝杆菌中MycP1蛋白(MycP124-422)的晶体结构,分辨率为2.15A。结构表明MycP1具有典型的类似subtilisin的丝氨酸蛋白酶的折叠模式,包含标志性的催化三联体Asp-His-Ser。但与subtilisin家族的丝氨酸蛋白酶不同,MycP1蛋白氨基端被定义为propeptide的序列是无序结构,并且不能被蛋白自身切除。为了进一步研究propeptide在MycP1蛋白结构与功能中的作用,我们表达并纯化了切除这一序列的MycP1蛋白(MycP163-422),并解析了它的晶体结构,分辨率为2.25A。MycP124-422和MycP163-422在三维结构上并没有显著差别,蛋白核心结构域的构象也不受propeptide切除的影响。相关生化实验结果表明,propeptide不会抑制核心结构域的活性,相反的,该序列的缺失反而导致MycP1的蛋白酶活性略微下降。上述研究结果表明,MycP1蛋白的‘"propeptide"序列并不具有经典的propeptide的结构与功能,并不符合经典的propepide的定义。分子动力学模拟的结果表明,MycP1蛋白的"propeptide"在功能上具有维持MycP1蛋白核心结构域构象稳定,从而维持其蛋白酶活性的作用。
本文的第二部分是结核分枝杆菌外膜蛋白Rv1698的结构研究。结核分枝杆菌具有复杂的包膜结构,通透性极弱,是保护细菌的一道天然屏障,也是致病性所必须的。在同样具有包膜结构的革兰氏阴性菌中,外膜蛋白行使着细菌跨越包膜与外界物质交换的功能。在结核分枝杆菌中,已鉴定了三个外膜蛋白——OmpATb、Rv1698和Rv1973;而在整个分枝杆菌家族中,目前只有结核分枝杆菌的外膜蛋白OmpATb和耻垢分枝杆菌的外膜蛋白MspA的结构被解析出来。对于Rv1698蛋白,研究表明它与结核分枝杆菌的致病性有密切关系,参与细菌对外界营养物质的摄取,是抗生素进入细菌胞内的重要通道。也有研究认为Rv1698蛋白可能参与结核分枝杆菌体内铜离子的外排。我们运用X-射线晶体衍射方法解析了Rv1698蛋白截短体(Rv169827-314)分别在含有和不含去垢剂C12E8的条件下的晶体结构,分辨率分别为2.3A和3.15A。晶体结构显示,Rv169827-314蛋白并不具有经典β-barrel通道结构,而是α/β结构模式,包含一个羧基端的球形结构域和一个氨基端长约70A、向外延伸的α-螺旋。相关生物化学和生理学实验表明,预测为信号肽的氨基端跨膜序列在生理状态下的成熟蛋白中并未被切除,而且对于Rv1698蛋白的膜定位和多聚化有重要作用,在多聚化的Rv1698蛋白中可以检测到通道活性。我们认为Rvl698蛋白可能通过自身的多聚化来形成通道结构,氨基端跨膜螺旋是影响蛋白多聚化的一个关键因素,并且参与通道的形成。Rv1698具有特殊的结构模式,可能代表着结核分枝杆菌外膜上一类新的通道蛋白结构,但这类蛋白的通透性特点和机制仍有待进一步研究。
Tuberculosis (TB) is one of the most common causes of death globally. Mycobacterium tuberculosis, the etiological agent of TB, still remains to emerge as a major public health threat because of the high pathogenicity, a significant increase in multiple-drug-resistance and the ability of immune evasion. As a progress of studies in pathogenicity and multiple-drug-resistance of M. tuberculosis, a number of related proteins have been identified and characterized. This dissereation focuses on the regulatory protein MycPl of mycobacterial virulence factor secretion system ESX-1and the outer-membrane prion Rv1698in M. tuberculosis. The structures of these proteins are determined using X-ray diffraction method, and the releationship between structure and function is investigated. These work could be helpful to explain the molecular etiology of M. tuberculosis, which can provide the the theoretical foundation to the prevention and treatment of TB.
In Chapter2, we report structural and functional studies of mycobacterial MycP1. In mycobacterium, a secretion system different from the known Type Ⅰ to Type Ⅵ secretion systems in Gram-negative bacteria was found and defined as Type Ⅶ secretion system (T7SS). The ESX-1system, a typical T7SS in M. tuberculosis, is the most extensively studied secretion system so far. M. tuberculosis use the ESX-1secretion system to deliver virulence proteins during infection. The ESX-1secretion system, therefor, was identified as a key virulence determinant in M. tuberculosis. The identity of all ESX-1substrates and the mechanism by which they affect host cells are not well understood. Various activities have been ascribed to the ESX-1substrates ESAT-6and CFP-10, including inhibition of phagosome maturation and cytokine signaling by infected macrophages, interaction with the macrophage immune receptor TLR2and inhibition of TLR signaling, and formation of pores in mycobacterial phagosomes, perhaps allowing bacterial spread. The regulation mechanism of ESX-1secretion system is an increasing concern. Ohol, Y. M. et al reported a mechanism of posttranscriptional control of ESX-1mediated by MycPl, a putative subtilisin-like serine protease with a C-terminal transmembrane helix. MycPl plays a dual role in regulating secretion activity of the ESX-1system. MycPl protein is required for the integrity and secretion activity of the ESX-1system. A mycPl deletion mutant in M. tuberculosis leads to the loss of the secretion activity for ESAT-6/CFP-10. Whereas the MycP1protein is required for secretion, abolition of MycP1protease activity by mutagenesis of the active site leads to increased secretion. Further more, EspB protein was identified as a substrate of MycPl. It was concluded that the MycP1protein is required for ESX-1secretion but that its protease activity negatively regulates secretion via EspB. As a subtilisin-like serine protease, MycP1contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser, classic hallmarks of a subtilase family serine protease. The MycP1propeptide was previously reported to be initially inactive and activated after prolonged incubation. In this study, we determined crystal structures of MycP1with (MycP124-422) and without (MycP163-422) the propeptide, and conducted EspB cleavage assays using the two proteins. Very high structural similarity was observed in the two crystal structures. Interestingly, protease assays demonstrated positive EspB cleavage for both proteins, indicating that the putative propeptide does not inhibit protease activity. Molecular dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP124-422than in MycP163-422, suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.
Chapter3presents our work on the outer-membrane protein Rv1698from M. tuberculosis. It has been reported that mycobacteria has a complex cell wall, which forms an exceptionally strong permeability barrier because of its extremely low permeability. The protective function of cell wall is an essential virulence factor of M. tuberculosis and contributes to its intrinsic drug resistance. Cryo-electron microscopy showed that mycobacterial cell walls include an unusual outer membrane, which composes of long-chain mycolic acids and a large variety of other extractable lipids. Meanwhile, a numer of proteins were provided to locate in the outer-membrane. In Gram-negative bacteria, the outer-membrane proteins contributes to the transport processes across the outer membrane. Escherichia coli use more than60proteins to functionalize its outer membrane. While only three mycobacterial outer membrane proteins (OMPs)(OmpATb, Rv1973and Rv1698) are known in M. tuberculosis so far. Bioinformatics analysis by Song, H. et al revealed that Rv1698is an outer-membrane protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Expression of rv1698restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. Lipid bilayer experiments demonstrated that purified Rv1698is an integral membrane protein that indeed produces channels. Taken together, these experiments demonstrated that Rv1698is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. In our work, crystals of Rv169827-314(with truncation of the N-terminal signal peptide) in detergent-containing buffer and aqueous solution were collected and diffracted at the resolutions of2.30and3.25A, respectively. The two crystal structures were in high similarity, indicating no pronounced conformational differences were induced by the presence of detergent. A mixed α/β-globular structure with a long helix extending away from the globular domain was observed for Rv169827-314, rather than a previously proposed integral membrane proteins, neither a beta-barrel porin like structure nor multi-span helical structure. It was also found that the predicted N-terminal signal sequence is not removed in the mature protein, and plays an improtant role in the oliogmerzation and membrane location of Rvl698. The cryo-EM showed that the purified full-length Rv1698protein forms hexamer in the presence of LDAO. An overall architecture model of Rv1698hexamer was built. The reported structures of Rv169827-314and the overall architecture model of the protein suggest alternative modes of membrane association, demanding further investigations combining structural biology and bacterial physiology.
本论文的第一部分是分枝杆菌ESX-1分泌系统调节蛋白MycP1的结构生物学研究。结核分枝杆菌中存在一种新型分泌系统T7SS (Type Ⅶ secretion system,), ESX-1系统就是一种典型的T7SS,也是分枝杆菌中研究最广泛的分泌系统。ESX-1分泌系统是分枝杆菌致病性的决定因素之一,致病性分枝杆菌在感染宿主细胞的过程中会通过ESX-1系统分泌致病性蛋白ESAT-6和CFP-10,通过抑制巨噬细胞内吞噬小体的成熟和细胞因子信号、抑制Toll样受体信号通路、在吞噬小体表面形成通道以促进细菌扩散等作用机制促进病菌侵染过程。MycP1是ESX-1系统的组成蛋白之一,对ESX-1系统有双重调控作用：一方面MycPl是维持ESX-1系统完整性所必须的,它的缺失会导致ESX-1系统丧失对ESAT-6/CFP-10的分泌功能；另一方面,MycP1对ESX-1系统的活性具有负调控作用,MycP1活性的缺失可以促进ESX-1系统对ESAT-6/CFP-10的分泌。MycP1的双重调节作用的意义可能在于维持分枝杆菌致病性与免疫原性之间的平衡,避免细菌在侵染宿主细胞的过程中激发宿主过强免疫反应。实际上,MycP1是一种subtilisin-like的丝氨酸蛋白酶,具有跨膜结构,研究证实MycP1对ESX-1系统的负调控是通过降解其天然底物EspB实现的。我们所感兴趣的是,MycP1蛋白作为一个具有双重调控作用的丝氨酸蛋白酶,它在结构上有没有特殊性,在功能上与EspB蛋白相互作用的机制是什么,它又是如何行使双重调控功能的。我们成功地解析了耻垢分枝杆菌中MycP1蛋白(MycP124-422)的晶体结构,分辨率为2.15A。结构表明MycP1具有典型的类似subtilisin的丝氨酸蛋白酶的折叠模式,包含标志性的催化三联体Asp-His-Ser。但与subtilisin家族的丝氨酸蛋白酶不同,MycP1蛋白氨基端被定义为propeptide的序列是无序结构,并且不能被蛋白自身切除。为了进一步研究propeptide在MycP1蛋白结构与功能中的作用,我们表达并纯化了切除这一序列的MycP1蛋白(MycP163-422),并解析了它的晶体结构,分辨率为2.25A。MycP124-422和MycP163-422在三维结构上并没有显著差别,蛋白核心结构域的构象也不受propeptide切除的影响。相关生化实验结果表明,propeptide不会抑制核心结构域的活性,相反的,该序列的缺失反而导致MycP1的蛋白酶活性略微下降。上述研究结果表明,MycP1蛋白的‘"propeptide"序列并不具有经典的propeptide的结构与功能,并不符合经典的propepide的定义。分子动力学模拟的结果表明,MycP1蛋白的"propeptide"在功能上具有维持MycP1蛋白核心结构域构象稳定,从而维持其蛋白酶活性的作用。
本文的第二部分是结核分枝杆菌外膜蛋白Rv1698的结构研究。结核分枝杆菌具有复杂的包膜结构,通透性极弱,是保护细菌的一道天然屏障,也是致病性所必须的。在同样具有包膜结构的革兰氏阴性菌中,外膜蛋白行使着细菌跨越包膜与外界物质交换的功能。在结核分枝杆菌中,已鉴定了三个外膜蛋白——OmpATb、Rv1698和Rv1973;而在整个分枝杆菌家族中,目前只有结核分枝杆菌的外膜蛋白OmpATb和耻垢分枝杆菌的外膜蛋白MspA的结构被解析出来。对于Rv1698蛋白,研究表明它与结核分枝杆菌的致病性有密切关系,参与细菌对外界营养物质的摄取,是抗生素进入细菌胞内的重要通道。也有研究认为Rv1698蛋白可能参与结核分枝杆菌体内铜离子的外排。我们运用X-射线晶体衍射方法解析了Rv1698蛋白截短体(Rv169827-314)分别在含有和不含去垢剂C12E8的条件下的晶体结构,分辨率分别为2.3A和3.15A。晶体结构显示,Rv169827-314蛋白并不具有经典β-barrel通道结构,而是α/β结构模式,包含一个羧基端的球形结构域和一个氨基端长约70A、向外延伸的α-螺旋。相关生物化学和生理学实验表明,预测为信号肽的氨基端跨膜序列在生理状态下的成熟蛋白中并未被切除,而且对于Rv1698蛋白的膜定位和多聚化有重要作用,在多聚化的Rv1698蛋白中可以检测到通道活性。我们认为Rvl698蛋白可能通过自身的多聚化来形成通道结构,氨基端跨膜螺旋是影响蛋白多聚化的一个关键因素,并且参与通道的形成。Rv1698具有特殊的结构模式,可能代表着结核分枝杆菌外膜上一类新的通道蛋白结构,但这类蛋白的通透性特点和机制仍有待进一步研究。
Tuberculosis (TB) is one of the most common causes of death globally. Mycobacterium tuberculosis, the etiological agent of TB, still remains to emerge as a major public health threat because of the high pathogenicity, a significant increase in multiple-drug-resistance and the ability of immune evasion. As a progress of studies in pathogenicity and multiple-drug-resistance of M. tuberculosis, a number of related proteins have been identified and characterized. This dissereation focuses on the regulatory protein MycPl of mycobacterial virulence factor secretion system ESX-1and the outer-membrane prion Rv1698in M. tuberculosis. The structures of these proteins are determined using X-ray diffraction method, and the releationship between structure and function is investigated. These work could be helpful to explain the molecular etiology of M. tuberculosis, which can provide the the theoretical foundation to the prevention and treatment of TB.
In Chapter2, we report structural and functional studies of mycobacterial MycP1. In mycobacterium, a secretion system different from the known Type Ⅰ to Type Ⅵ secretion systems in Gram-negative bacteria was found and defined as Type Ⅶ secretion system (T7SS). The ESX-1system, a typical T7SS in M. tuberculosis, is the most extensively studied secretion system so far. M. tuberculosis use the ESX-1secretion system to deliver virulence proteins during infection. The ESX-1secretion system, therefor, was identified as a key virulence determinant in M. tuberculosis. The identity of all ESX-1substrates and the mechanism by which they affect host cells are not well understood. Various activities have been ascribed to the ESX-1substrates ESAT-6and CFP-10, including inhibition of phagosome maturation and cytokine signaling by infected macrophages, interaction with the macrophage immune receptor TLR2and inhibition of TLR signaling, and formation of pores in mycobacterial phagosomes, perhaps allowing bacterial spread. The regulation mechanism of ESX-1secretion system is an increasing concern. Ohol, Y. M. et al reported a mechanism of posttranscriptional control of ESX-1mediated by MycPl, a putative subtilisin-like serine protease with a C-terminal transmembrane helix. MycPl plays a dual role in regulating secretion activity of the ESX-1system. MycPl protein is required for the integrity and secretion activity of the ESX-1system. A mycPl deletion mutant in M. tuberculosis leads to the loss of the secretion activity for ESAT-6/CFP-10. Whereas the MycP1protein is required for secretion, abolition of MycP1protease activity by mutagenesis of the active site leads to increased secretion. Further more, EspB protein was identified as a substrate of MycPl. It was concluded that the MycP1protein is required for ESX-1secretion but that its protease activity negatively regulates secretion via EspB. As a subtilisin-like serine protease, MycP1contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser, classic hallmarks of a subtilase family serine protease. The MycP1propeptide was previously reported to be initially inactive and activated after prolonged incubation. In this study, we determined crystal structures of MycP1with (MycP124-422) and without (MycP163-422) the propeptide, and conducted EspB cleavage assays using the two proteins. Very high structural similarity was observed in the two crystal structures. Interestingly, protease assays demonstrated positive EspB cleavage for both proteins, indicating that the putative propeptide does not inhibit protease activity. Molecular dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP124-422than in MycP163-422, suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.
Chapter3presents our work on the outer-membrane protein Rv1698from M. tuberculosis. It has been reported that mycobacteria has a complex cell wall, which forms an exceptionally strong permeability barrier because of its extremely low permeability. The protective function of cell wall is an essential virulence factor of M. tuberculosis and contributes to its intrinsic drug resistance. Cryo-electron microscopy showed that mycobacterial cell walls include an unusual outer membrane, which composes of long-chain mycolic acids and a large variety of other extractable lipids. Meanwhile, a numer of proteins were provided to locate in the outer-membrane. In Gram-negative bacteria, the outer-membrane proteins contributes to the transport processes across the outer membrane. Escherichia coli use more than60proteins to functionalize its outer membrane. While only three mycobacterial outer membrane proteins (OMPs)(OmpATb, Rv1973and Rv1698) are known in M. tuberculosis so far. Bioinformatics analysis by Song, H. et al revealed that Rv1698is an outer-membrane protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Expression of rv1698restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. Lipid bilayer experiments demonstrated that purified Rv1698is an integral membrane protein that indeed produces channels. Taken together, these experiments demonstrated that Rv1698is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. In our work, crystals of Rv169827-314(with truncation of the N-terminal signal peptide) in detergent-containing buffer and aqueous solution were collected and diffracted at the resolutions of2.30and3.25A, respectively. The two crystal structures were in high similarity, indicating no pronounced conformational differences were induced by the presence of detergent. A mixed α/β-globular structure with a long helix extending away from the globular domain was observed for Rv169827-314, rather than a previously proposed integral membrane proteins, neither a beta-barrel porin like structure nor multi-span helical structure. It was also found that the predicted N-terminal signal sequence is not removed in the mature protein, and plays an improtant role in the oliogmerzation and membrane location of Rvl698. The cryo-EM showed that the purified full-length Rv1698protein forms hexamer in the presence of LDAO. An overall architecture model of Rv1698hexamer was built. The reported structures of Rv169827-314and the overall architecture model of the protein suggest alternative modes of membrane association, demanding further investigations combining structural biology and bacterial physiology.
引文
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